Antibody-targeted chromatin enables effective intracellular delivery and functionality of CRISPR/Cas9 expression plasmids

Abstract

We report a novel system for efficient and specific targeted delivery of large nucleic acids to and into cells. Plasmid DNA and core histones were assembled to chromatin by salt gradient dialysis and subsequently connected to bispecific antibody derivatives (bsAbs) via a nucleic acid binding peptide bridge. The resulting reconstituted vehicles termed 'plasmid-chromatin' deliver packaged nucleic acids to and into cells expressing antigens that are recognized by the bsAb, enabling intracellular functionality without detectable cytotoxicity. High efficiency of intracellular nucleic acid delivery is revealed by intracellular expression of plasmid encoded genes in most (∼90%) target cells to which the vehicles were applied under normal growth/medium conditions in nanomolar concentrations. Specific targeting, uptake and transgene expression depends on antibody-mediated cell surface binding: plasmid chromatin of identical composition but with non-targeting bsAbs or without bsAbs is ineffective. Examples that demonstrate applicability, specificity and efficacy of antibody-targeted plasmid chromatin include reporter gene constructs as well as plasmids that enable CRISPR/Cas9 mediated genome editing of target cells.

Figure 1.

MNase digestion of antibody–chromatin complexes and antibody-complexation with plasmid-chromatin. (A) Agarose gel electrophoresis of chromatin without (lane 1–3) and in presence of biotin-CPXM2 ∼ anti biotin 2+2 bsAb constructs (lane 4–6) after partial DNA hydrolysis by MNase with increasing incubation time (20s, 80s and 270s); Chromatin without MNase treatment (lane 7) is shown as control. Mononucleosomal DNA bands (147bp) indicate complete digestion in contrast to higher molecular weight bands. In presence of biotin-CPXM2 ∼ anti biotin 2+2 bsAb constructs, chromatin is more nuclease resistant as the 147 bp DNA band only occurs at late time-points of Nuclease treatment in comparison to the chromatin only sample. (B) Scheme of antibody–chromatin complexes with plasmid DNA reconstituted into nucleosomes and associated antibody-peptide constructs. Variable regions against cell surface antigen (blue) faces outwards and anti biotin scFv (green) is bound at biotin-CPXM2 peptide (purple) associated at the DNA backbone. (C) MST runs for Chromatin + biotin-CPXM2 ∼ anti biotin 2+2 bsAb interaction. Ligand concentration refers to biotin–CPXM2 peptide (twice as much as the respective anti biotin 2+2 bsAb concentration). Exp 1 (blue) and Exp 2 (red) are independent experiments of the same construct with the respective curve fit for K_D determination.

Figure 2.

Flow cytometric determination of delivery specificity; Binding and uptake of antibody-Cy3 and DNA-Cy5 (before and after chromatin assembly) was analysed by flow cytometry after incubation for 1 h. (A) Histogram of MCF7 cells after treatment with targeted (anti LeY; dotted red) and untargeted (anti CD33; dotted blue) DNA-Cy5 complexes. Cy5 signal was detected only after treatment with the targeted DNA-Cy5 construct. (B) Histogram of MCF7 cells after treatment with targeted (red) and untargeted (blue) chromatin-Cy5 complexes. Results are comparable to results after DNA–Cy5 delivery. (C) Contours plot of MCF7 Cy3 (x-axis) and Cy5 (y-axis) signals after treatment with various antibody chromatin complexes comprising Cy3 labelled antibody and Cy5 labelled DNA. Cells were treated with complexes comprising antibody without specificity against cell surface antigen but against CPXM2 peptide do neither show Cy3 nor Cy5 signals (blue). Cells treated with complexes comprising digoxigenin CPXM2 peptide (instead of biotin CPXM2 peptide) display Cy3 signal but no Cy5 signal, demonstrating that antibody but not chromatin is present at the cell surface (green). Cells treated with complexes comprising antibody with specificity against the cell surface and CPXM2 peptide display Cy3 signal and Cy5 signal, demonstrating that antibody as well as chromatin is present at the cell surface (red).

Figure 3.

Efficiency and cytotoxicity of gene delivery systems. (A) Delivery efficiency of EGFP expression plasmids was addressed by determination of GFP positive cells via flow cytometry 48 h after treatment with targeting complexes being present throughout that time. Significant numbers of GFP positive cells were achieved with Lipofection and anti LeY-chromatin complexes for MCF-7 cells. (B) Cytotoxicity was addressed by quantification of LDH release. Significant LDH release after 48 h was only observed with lipofection. Cells were exposed to respective treatment for the whole incubation period in normal (serum containing) cell culture medium. Mean values + SEM are shown (n = 3); P-values < 0.001 are indicated by three asterisks.

Figure 4.

Confocal microscopy analysis of antibody mediated plasmid DNA or chromatin delivery and intracellular routing. (A) Live cell imaging of MCF7 cells 4h after treatment with targeted (LeY) chromatin (top row), targeted (LeY) DNA (middle row) and untargeted (CD33) chromatin (bottom row); Antibody-Cy3 signal is displayed in green and DNA-Cy5 level is displayed in red; Overlay of fluorescent images are shown in the ‘Merge’ column and the right column shows respective transmission images. (B) Live cell imaging of MCF7 cells 3 days after treatment with targeted (LeY) chromatin complexes comprising unlabelled antibody and Cy5 labelled DNA. Left panel shows GFP signal in green, middle panel DNA–Cy5 in cyan and right panel transmission, respectively. (C) Imaging of fixed MCF7 cells 3 days after treatment with targeted (LeY) chromatin. Left panel displays DNA Cy5 signal in pseudocolor, middle panel shows antibody signal generated by counterstaining with anti human IgG Cy3 antibody in red and the right panel represents the transmission image. Cell surface, vesicular compartments and nuclear envelope are marked by yellow contours. Scale bars: 50 μm.

Figure 5.

Antibody-chromatin delivery with plasmid DNA encoding a CRISPR/Cas9 system. (A) Agarose gel electrophoresis of DPH1 gRNA/Cas9 plasmid DNA (lanes 1–3) and chromatin (lanes 4–6) after partial DNA hydrolysis by MNase with increasing incubation time (20s, 80s and 270s). (B) Representative microscopic images of DT resistant MCF7 cell clones after treatment with vehicle (PBS control), targeted control plasmid chromatin (LeY-Chromatin (eGFP)), non-targeted Cas9/DPH1 gRNA plasmid chromatin (CD33-Chromatin (Cas9/DPH1 gRNA)), targeted Cas9/DPH1 gRNA plasmid chromatin (LeY-Chromatin (Cas9/DPH1 gRNA)); DT resistant colonies were only observed after treatment with targeted DPH1 gRNA/Cas9 plasmid chromatin.